Fatty acid transport proteins (FATPs) are a family of integral membrane proteins, several of which function in the transport and activation of exogenous fatty acids across the plasma membrane. FATP2 is expressed in the intestine and liver, yet its physiological role in normal fatty acid homeostasis in these cells types has not been defined. We have identified two splice variants of human FATP2, which exhibit distinguishing biochemical activities. The first form (FATP2a), also described as a very long chain acyl CoA synthetase has a Mr of 70,000 while the second form (FATP2b) obtained from the Image Consortium library has a Mr of 65,000. FATP2b lacks exon3, which encodes a region of the protein that contains part of the ATP binding domain and required for adenylate formation. Both forms of FATP2 are proficient in fatty acid transport yet FATP2b is unable to activate very long chain fatty acids. These studies thus define two naturally occurring variants of a member of the FATP family that distinguish their roles in fatty acid transport and activation. Using 293 T-REx cells, which allows regulated expression of FATP2a and FATP2b, experiments will be completed to [1] define their individual roles in fatty acid transport and activation and [2] address whether they direct fatty acids into different metabolic pools as assessed using stable isotopically-labeled fatty acids in combination with high resolution mass spectrometry. To access the physiological roles of FATP2 in the intestine and liver, shRNA will be used to knockdown FATP2 expression in Caco-2 (intestinal like) and HepG2 (hepatic like) cells, and patterns of fatty acid transport, activation and intracellular trafficking assessed. The individual roles of these two variants will be further assessed using five small molecule inhibitors selected from high throughput screens for small compounds that disrupt fatty acid transport. Collectively, these studies are expected to provide novel insights into the differential roles of FATP2a and FATP2b in fatty acid homeostasis. Dislipidemia has been shown to be a contributing factor to metabolic diseases such a diabetes, obesity, and cardiovascular disease. Characterizing the proteins involved in lipid metabolism can help elucidate the biological processes which govern these pathological states. The focus of this research proposal includes characterizing the function of FATP2 which is expected to give insight into the biological processes underpining lipid metabolism.